The objective of this research is to identify and characterize the hierarchy of regulatory phenomena which govern the expression of a simple metabolic pathway in a eukaryotic organism. The pathway is that of nitrate assimilation in Neurospora crassa which consists of two metalloflavoenzymes: nitrate reductase and nitrite reductase. Both enzymes of this pathway are induced by either nitrate or nitrite and both are repressed by the end product, ammonia. The latter effect is dominant. Through the use of specific immunoelectrophoretic techniques, the kinetics of synthesis and degradation of the nitrate reductase will be determined under varying nutritional conditions. In addition, nitrate reductase is rapidly and specifically inactivated in vivo. An immediate goal of this project is the elucidation of the mechanism which governs this inactivation. Clues to this mechanism will be sought by examining the extent of alteration in the structural integrity of the nitrate reductase upon in vivo inactivation. The isolation and characterization of the inactivation system in vitro will then be undertaken. Within the overall objective, other in vitro studies will be conducted to determine whether the activities of the nitrate and nitrite reductase are modulated through interaction with metabolities, covalent modification and/or changes in the relative oxidation-reduction states of the enzymes. Together, these results should provide an integrated scheme describing the controls exerted on this selected system. Such a scheme would be of general value, through analogy, to related problems of metabolic regulation in eukaryotes. BIBLIGRAPHIC REFERENCE: Amy, N.K., and R.H. Garrett. "Immunoelectrophoretic Determination of Nitrate Reductase in the nit Mutants of Neurospora crassa." American Society of Microbiology Abstracts 77: 204 (1977).